Reflecting cell strains, development and differentiation stages, pathologic conditions, a sugar chain having each characteristic structure is expressed in mammalian cells. Sugar chains exist in a cell surface layer and a serum in the form of a complex carbohydrate such as a glycoprotein and a glycolipid, and an effective utilization as a biomarker has been examined from the properties in the same manner as a protein and nucleic acid. In recent years, it begins to find out that sugar chains has an important function in vivo, and researches relating to the function have been advanced.
However, a functional analysis has scarcely progressed since techniques relating to structural identification and detection of a sugar chain are not sufficiently established as compared to protein and nucleic acid. If an antibody capable of distinguishing and detecting a partial structure of a sugar chain, which characterizes a sugar chain structure of a complex carbohydrate, could be easily developed, a sugar chain analysis by ELISA, a western blot method, etc., which are generally employed in a protein analysis, is possible, and research developments relating to functional clarification and industrial applications of sugar chains are considered to be accelerated.
Among sugar chain structures of complex carbohydrates, particularly, an N-linked sugar chain structure contained in a glycoprotein becomes various cancer diagnosis markers or diagnosis markers for serious diseases in many cases; therefore, expectation of development of an antibody that can recognize an N-linked sugar chain structure is significant.
For example, a change in an N-linked sugar chain structure of a glycoprotein in liver cancer has been known for a use as a diagnosis marker so far (Non Patent Literature 1). A typical example thereof is an N-linked sugar chain of α-fetoprotein (AFP). An increase of a serum value of AFP has been utilized as a diagnostic index of liver cancer for a long time; however, there was a defect such that only liver cancer cannot be precisely diagnosed since the serum value of AFP increases also in hepatitis and hepatic cirrhosis. It was recently found that an N-linked sugar chain structure of an AFP protein is chained specifically in liver cancer (Non Patent Literature 2), and AFP-L3 containing such a sugar chain structure is focused as a highly precise diagnosis marker for liver cancer. Regarding ovarian cancer, immunoglobulins and acute-phase proteins, which contain core fucose type and sLeX type N-linked sugar chains, have been found as novel marker candidates (Non Patent Literature 3).
In addition, an N-linked sugar chain started finding to be useful as a marker of hepatic fibrosis (previous stage of hepatic cirrhosis) which is a previous symptom of hepatic cirrhosis. An N-linked sugar chain of an α1-acid glycoprotein (AGP) existing in the serum has a characteristic structure correlating to the onset of hepatic fibrosis, and LecT-Hepa has been developed as a marker that diagnoses the onset of hepatic fibrosis (Non Patent Literature 4). What is more, by FastLec-Hepa that has been improved so as to be able to detect at high sensitivity and with simplicity, a change specific to hepatic fibrosis, which is found in an N-linked sugar chain of an M2BP glycoprotein in the blood, is detected (Non Patent Literature 5).
Other than the above, abnormality of an N-linked sugar chain structure of a glucose transporter protein, which is expressed in the pancreas by diabetes, and the like, has been proposed to be a cause of developing the pathologic condition (Non Patent Literature 6).
From the background as described above, an expectation of a technique of easily preparing an antibody that can recognize a sugar chain structure of a complex carbohydrate, particularly, an N-linked sugar chain structure in a glycoprotein has more increased; however, when an oligosaccharide chain constituting a specific partial structure of a sugar chain part in a complex carbohydrate is used as an immunogen, it is very hard to obtain a practical and useful antibody. The greatest reason is because, in the immune system of a mammalian body utilized as a host producing an antibody, development of the immune system which recognizes an oligosaccharide chain as an antigen is insufficient. In particular, production of an antibody which recognizes “an N-linked sugar chain-derived oligosaccharide chain in a glycoprotein” has been known to be difficult (Non Patent Literatures 7 and 8), and an effective antibody is not hardly developed even to the present date.
On the other hand, when a peptide is an immunogen, there is a technique of utilizing an immunity induction system by antigen presentation by use of an MHC molecule and enhancing the immunogenicity. In the method, any peptide is bound to a carrier protein such as KLH and BSA to form an immunity inducer, thereby enhancing an interaction with the MHC molecule through a carrier protein, and an objective antigen can be a thymus-dependent antigen. When the objective antigen can be a thymus-dependent antigen, a B cell that is an antibody producing cell is capable of production of a useful antibody with high specificity and broad application by undergoing processes such as class switching, affinity maturation, and memory of antibody production due to interaction with a helper T cell.
Also when an oligosaccharide chain is an immunogen, there is a report that a conjugate (artificial glycoprotein) with a carrier protein such as KLH and BSA, which are used to the peptide, was prepared and immunized to produce an IgG antibody recognizing a target carbohydrate antigen (Non Patent Literatures 9 and 10). However, the oligosaccharide chain in the report is an examination of using a polymer of α1,6 glucose (derived product of isomaltose) prepared by dextran derived from microorganisms, which is not an examination of using a mammalian complex carbohydrate-derived oligosaccharide chain, at the same time, the affinity of the obtained monoclonal antibody is low as an association constant of approximately 102 M−1 to 105 M−1 (Non Patent Literature 11), and the examination was not sufficient as a production method of development of a monoclonal antibody. Also from this result, even when the same conjugation method in peptide immunization is directly applied in the case of a mammalian cell-derived oligosaccharide chain, a possibility of functioning as a thymus-dependent antigen is low, an antibody finally showing sufficient affinity and specificity is thinly estimated and, thus, this technique could not be a practical option for a person skilled in the art. Therefore, a research of a carrier compound as an alternative to the technique has been activated.
A method of inducing immunity by using, as an immunogen, a material obtained by reductively bonding an oligosaccharide chain to an amino group in biotinylated aminopyridine and subsequently polyvalently bonding biotin to avidin that is a ligand (Non Patent Literature 12) has been known so far, and although immunity is induced using the above described isomaltose derived product (heptasaccharide) as an oligosaccharide chain, immunity enhancement ability is low and development of an effective monoclonal antibody to a target oligosaccharide chain is not attained.
In addition, regarding “Galβ1,4GlcNAcβ1,2Man”, “a β-GlcNAc residue in a nonreducing end” and “Siaα2,6Galβ1,4GlcNAc (CDw75)”, which are oligosaccharide antigens in N-linked sugar chains of typical glycoproteins, a method of inducing immunity by using phosphatidyl ethanolamine as a carrier compound and using a compound obtained by reductively bonding an oligosaccharide chain to the amino group as an immunogen (Non Patent Literatures 7 and 8) has been reported. However, affinity and specificity are insufficient in monoclonal antibodies obtained by these immunity inducing methods, a practical antibody therefore cannot be considered to be provided and the immunity inducing methods are not generally used techniques of selectively inducing an antibody to any sugar chain. What is more, in these immunity inducing methods, since a reductive amination method of cleaving the reducing end sugar of an oligosaccharide chain is utilized as a production method in conjugating a carrier compound and the oligosaccharide chain, an immunity inducer cannot be synthesized with keeping the structure of the oligosaccharide chain. Therefore, the methods have a defect such that a spacer is essential in order to prevent cleavage and a complicated synthesis process of previously bonding sugar or hydroxybenzaldehyde for a spacer to an oligosaccharide chain is required.
In addition, a method of using magnetic metallic nano particles that are obtained by bonding to a sugar chain ligand with an adjuvant through a linker (Patent Literature 1) has been proposed, but preparation of nano particles is not easy, and a process of bonding a sugar chain ligand and an adjuvant to the nano particles at a constant ratio is also not easy. It is proposed that, to a Globo-H carbohydrate specific to breast cancer, a carrier protein that is a diphtheria toxin cross-reacting substance is bound to the reducing side of the Globo-H carbohydrate through aminoalkyl and p-nitrophenyl ester to form a cancer vaccine (Patent Literature 2), which however is not a generally applicable technique that can apply to a general carbohydrate antigen.
According to the above description, desired was development of a carrier compound which becomes an excellent immunity inducer applicable to widely general oligosaccharide antigens, in particular, a glycoprotein N-linked sugar chain-derived oligosaccharide chain that is hardly produced and is capable of induction of an anti-carbohydrate antigen monoclonal antibody with high specificity and affinity. In addition, a production method capable of easily bonding a carrier compound and an oligosaccharide chain with keeping the structure of the target oligosaccharide antigen was also desired.